Difference between revisions of "Part:BBa K4800008"
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<p style="font-size: 180%; font-weight: bold;">Test: | <p style="font-size: 180%; font-weight: bold;">Test: | ||
<p>PRSFDuet-YciA-sfp-Q302E-YahK and PRSFDuet-YciA-sfp-Q302E-SpyTag-YahK-EutM-SpyCatcher were respectively transfected into BL21 (DE3) and whole-cell catalyzed, cultured at 30°C 200 rpm for 5 h. Samples were taken at 1-h intervals, and the content of 1,5-PDO was detected by HPLC. </p > | <p>PRSFDuet-YciA-sfp-Q302E-YahK and PRSFDuet-YciA-sfp-Q302E-SpyTag-YahK-EutM-SpyCatcher were respectively transfected into BL21 (DE3) and whole-cell catalyzed, cultured at 30°C 200 rpm for 5 h. Samples were taken at 1-h intervals, and the content of 1,5-PDO was detected by HPLC. </p > | ||
− | <p><img src="https://static.igem.wiki/teams/4800/wiki/parts/prsfduet-ycia-sfp-q302e-spytag-yahk-eutm-spycatcher- | + | <p><img src="https://static.igem.wiki/teams/4800/wiki/parts/prsfduet-ycia-sfp-q302e-spytag-yahk-eutm-spycatcher-whole-cell.png" width="80%" height="80%"></p> |
<div align="center"> | <div align="center"> | ||
<strong>Fig2. Whole-cell catalytic results of KA30 vs KA30 (Q302E+EutM) </strong> | <strong>Fig2. Whole-cell catalytic results of KA30 vs KA30 (Q302E+EutM) </strong> |
Revision as of 18:54, 6 October 2023
Ptrc-sfp-MmCAR(Q302E)-SpyTag-EutM-SpyCatcher-YahK
Ptrc is the promoter, YciA-sfp-Q302E is a mutant gene derived from BBa_K1655000, EutM is the protein scaffold, Yahk is the aldo-keto reductase, and 'molecular glue' SpyTag/SpyCatcher can be used to construct multi-enzyme complexes.
Build:
The vector was linearized using PCR using PRSFDuet-YciA-sfp-Q302E-YahK as a template. The vector containing the SpyTag sequence was amplified by modifying the 5' end of the primer to amplify an EutM-SpyCatcher-containing fragment from a lab-conserved strain.In-fusion cloning will be used to link the fragment to the linearized vector. The recombinant plasmid will be transfected into the receptor cell Trans-T1 and coated on LB solid medium containing 100 μg/mL kanamycin, and incubated at 37°C for 12 h. Single colonies will be picked for colony PCR validation, and positive colonies will be selected for culture and sequencing to obtain the recombinant plasmid PRSFDuet-YciA-sfp-Q302E-SpyTag-YahK-EutM- SpyCatcher.
Test:
PRSFDuet-YciA-sfp-Q302E-YahK and PRSFDuet-YciA-sfp-Q302E-SpyTag-YahK-EutM-SpyCatcher were respectively transfected into BL21 (DE3) and whole-cell catalyzed, cultured at 30°C 200 rpm for 5 h. Samples were taken at 1-h intervals, and the content of 1,5-PDO was detected by HPLC.
By HPLC data, it was seen that BL21 (DE3) imported into PRSFDuet-YciA-sfp-Q302E-SpyTag-YahK-EutM-SpyCatcher catalyzed the production of more 1,5-PDO.
Electrically transfer PRSFDuet-YciA-sfp-Q302E-YahK+PTrc99a-davB-davA-GabT, PRSFDuet-YciA-sfp-Q302E-SpyTag-YahK-EutM-SpyCatcher+PTrc99a-davB-davA-GabT transferred to KA30 ∆YcjQ for fermentation. Samples were taken at 12h intervals for OD600, glucose and lysine content, and the fermentation broth was assayed for 1,5-PDO by HPLC.
By HPLC data, it was seen that KA30 fermentation imported into PRSFDuet-YciA-sfp-Q302E-SpyTag-YahK-EutM-SpyCatcher produced more 1,5-PDO.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1313
Illegal EcoRI site found at 5098
Illegal XbaI site found at 5016
Illegal SpeI site found at 527
Illegal PstI site found at 1748
Illegal PstI site found at 1775
Illegal PstI site found at 3044
Illegal PstI site found at 3071
Illegal PstI site found at 3464
Illegal PstI site found at 3960
Illegal PstI site found at 4604 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1313
Illegal EcoRI site found at 5098
Illegal NheI site found at 5786
Illegal SpeI site found at 527
Illegal PstI site found at 1748
Illegal PstI site found at 1775
Illegal PstI site found at 3044
Illegal PstI site found at 3071
Illegal PstI site found at 3464
Illegal PstI site found at 3960
Illegal PstI site found at 4604 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1313
Illegal EcoRI site found at 5098
Illegal BglII site found at 2302
Illegal BglII site found at 2928
Illegal BglII site found at 3204
Illegal BglII site found at 3895
Illegal BamHI site found at 1285
Illegal BamHI site found at 4859
Illegal BamHI site found at 5092
Illegal XhoI site found at 4844
Illegal XhoI site found at 4931 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1313
Illegal EcoRI site found at 5098
Illegal XbaI site found at 5016
Illegal SpeI site found at 527
Illegal PstI site found at 1748
Illegal PstI site found at 1775
Illegal PstI site found at 3044
Illegal PstI site found at 3071
Illegal PstI site found at 3464
Illegal PstI site found at 3960
Illegal PstI site found at 4604 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1313
Illegal EcoRI site found at 5098
Illegal XbaI site found at 5016
Illegal SpeI site found at 527
Illegal PstI site found at 1748
Illegal PstI site found at 1775
Illegal PstI site found at 3044
Illegal PstI site found at 3071
Illegal PstI site found at 3464
Illegal PstI site found at 3960
Illegal PstI site found at 4604
Illegal NgoMIV site found at 3572
Illegal NgoMIV site found at 3634
Illegal NgoMIV site found at 3851
Illegal NgoMIV site found at 4298
Illegal AgeI site found at 1478
Illegal AgeI site found at 2489
Illegal AgeI site found at 2786
Illegal AgeI site found at 3671
Illegal AgeI site found at 3680
Illegal AgeI site found at 4355 - 1000COMPATIBLE WITH RFC[1000]